Fibroblast growth factors (FGF) and their receptors (FGFR) play a central role in salivary gland branching morphogenesis. In mice, targeted disruption of Fgf10 or its receptor, Fgfr2b, causes salivary gland agenesis, demonstrating the essential role of FGF10-FGFR2b signaling pathways in salivary gland development. However, the lack of salivary glands in Fgf10 or Fgfr2b knockout mice precludes further investigation of branching morphogenesis in these mouse models. Therefore, it is necessary to develop an alternative approach to gain insights about the role of Fgfr2b intracellular signaling pathways in salivary gland branching morphogenesis. We have developed a novel mouse model system in which specific signaling pathways downstream of Fgfr2 have been abrogated by a knock-in mutation, without compromising the tyrosine kinase activity of the receptor to activate other signaling pathways. Our hypothesis is that Fgfr2b signaling via Frs21 is essential for salivary gland branching morphogenesis. In Aim 1, we will (1) analyze the branching morphogenesis of submandibular glands (SMGs) from different mutant mouse strains by histology and ex vivo organ culture; (2) use mesenchyme-free SMG cultures to investigate epithelial morphogenesis; and (3) use cell lines expressing the mutant and wild type receptors to evaluate the mechanism of epithelial transphosphorylation of Frs21 by Fgfr1b and Fgfr2b during branching morphogenesis. In Aim 2, we will determine the role of Frs21-mediated Grb2 and Shp2 signaling pathways in SMG branching morphogenesis. Our hypothesis is that Shp2 is the critical mediator of Fgfr2b signaling via Frs21 for SMG branching morphogenesis. We will test this hypothesis using two strains of genetically engineered mutant mice. In the first mutant mouse model, the four Grb2 binding sites are mutated to phenylalanine (4F), and thus cannot recruit Grb2. In the second mutant mouse model, the two Shp2 binding sites are mutated to phenylalanine (2F), and thus cannot recruit Shp2. We will perform (1) histology and ex vivo SMG organ culture to determine the role of Grb2 and Shp2 in branching morphogenesis; and (2) recombinant SMG culture to determine whether the mutations are cell autonomous or non-cell autonomous by culturing wild type epithelium with Grb2 or Shp2 mutant mesenchyme and vice versa. In addition, we propose two approaches to identify novel genetic pathways: a) genome-wide mRNA expression analysis in the epithelium and mesenchyme of the mutant SMGs, and b) expression-based pathway analysis of target genes in SMG. Collectively, this work will provide a detailed molecular picture of how FGF signaling, mediated by the Fgfr2b isoform and the docking protein Frs21, regulates salivary gland branching morphogenesis. The results of these studies may enable the design of novel methods for salivary gland regeneration using FGFR2b mediated pathways to regulate progenitor cell differentiation and morphogenesis. PUBLIC HEALTH RELEVANCE: Salivary gland dysfunction caused by mutations, radiotherapy or chemotherapy affect the quality of life of patients by causing oral dryness, dental caries, hampered speech, and xerostomia. The overall goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to Fgfr2b stimulation, specifically the Frs21-mediated pathways essential for the development of salivary glands. We anticipate that our findings will shed new light on fundamental intracellular signaling pathways essential for epithelial morphogenesis of salivary glands, and will enable the design of novel methods for salivary gland regeneration using FGFR2b-mediated pathways to regulate progenitor/stem cell differentiation and morphogenesis.